BACKGROUND: Milbemectin and abamectin are frequently used to control the spider 35 mite Tetranychus urticae. The development of abamectin resistance in this major pest 36 has become an increasing problem worldwide, potentially compromising the use of 37 milbemectin. In this study, a large collection of European field populations was 38 screened for milbemectin and abamectin resistance, allowing to thoroughly evaluate 39 any potential cross-resistance risk, and both target-site and metabolic resistance 40 mechanisms were investigated.41 RESULTS: High to very high levels of abamectin resistance were found in one third of 42 all populations, while milbemectin resistance levels were low for most populations. The 43 occurrence of well-known target-site resistance mutations in glutamate-gated chloride 44 channels (G314D in GluCl1 and G326E in GluCl3) was documented in the most 45 resistant populations. However, a new mutation, I321T in GluCl3, was also uncovered 46 in three resistant populations. A differential gene-expression analysis revealed the 47 overexpression of detoxification genes, more specifically cytochrome P450 48 monooxygenase (P450) and UDP-glycosyltransferase (UGT) genes. Subsequently, 49 multiple UGTs were functionally expressed, and their capability to glycosylate 50 abamectin and milbemectin, was tested and confirmed. 51 CONCLUSIONS: We found a clear correlation between abamectin and milbemectin 52 resistance in European populations of T. urticae, but as milbemectin resistance levels 53 were low, the observed cross-resistance is probably not of operational importance. The 54 presence of target-site resistance mutations in GluCl genes was confirmed in most but 55 not all resistant populations. Gene-expression analysis andr functional characterization 56 of P450s and UGTs suggests that also metabolic abamectin resistance mechanisms 57 are common in European T. urticae populations.

BACKGROUND The cys‐loop ligand‐gated ion channels, including the glutamate‐gated chloride channel (GluCl) and GABA‐gated chloride channel (Rdl) are important targets for drugs and pesticides. The macrocyclic lactone abamectin primarily targets GluCl and is commonly used to control the spider mite Tetranychus urticae, an economically important crop pest. However, abamectin resistance has been reported for multiple T. urticae populations worldwide, and in several cases was associated with the mutations G314D in GluCl1 and G326E in GluCl3. Recently, an additional I321T mutation in GluCl3 was identified in several abamectin resistant T. urticae field populations. Here, we aim to functionally validate this mutation and determine its phenotypic strength. RESULTS The GluCl3 I321T mutation was introgressed into a T. urticae susceptible background by marker‐assisted backcrossing, revealing contrasting results in phenotypic strength, ranging from almost none to 50‐fold. Next, we used CRISPR‐Cas9 to introduce I321T, G314D and G326E in the orthologous Drosophila GluCl. Genome modified flies expressing GluCl I321T were threefold less susceptible to abamectin, while CRISPRed GluCl G314D and G326E flies were lethal. Last, functional analysis in Xenopus oocytes revealed that the I321T mutation might reduce GluCl3 sensitivity to abamectin, but also suggested that all three T. urticae Rdls are affected by abamectin. CONCLUSION Three different techniques were used to characterize the role of I321T in GluCl3 in abamectin resistance and, combining all results, our analysis suggests that the I321T mutation has a complex role in abamectin resistance. Given the reported subtle effect, additional synergistic factors in resistance warrant more investigation. © 2020 Society of Chemical Industry

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Tetranychus urticae Koch is one of the most common and harmful pests in vegetable production areas. Similar to other countries, control of T. urticae is mainly based on acaricides in Turkey. However, T. urticae rapidly develops resistance and failures in chemical control have occurred frequently. The toxicity of various acaricides was investigated in 10 T. urticae populations collected from vegetable crops in Turkey. In addition, populations were screened for the presence of all currently known target-site resistance mutations. It was shown that resistance to bifenthrin was the most widespread, but also half of the populations were resistant to abamectin and hexythiazox. Resistance mutations in the voltage-gated sodium channel (VGSC) and chitin synthase 1 were found in various populations. Moreover, for the first time, F1538I and L1024V VGSC mutations were reported for Turkish populations. Mutations that confer resistance to abamectin, bifenazate and METI-I acaricides such as pyridaben were not detected. These results will contribute to the design of an effective resistance management program in Turkey.

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